The long-range goals of this project are delineation, at the molecular level, of: (a) the structural determinants responsible for specific, high-affinity binding of the heterodimeric glycoprotein hormone, human choriogonadotroin (hCG), to the lutropin/choriogonadotropin receptor (LHR), a member of the G protein-coupled receptor superfamily; (b) the mechanisms of ligand-dependent and ligand-independent LH receptor activation; and (c) the mechanism by which the receptor activates Gs to initiate intracellular signaling pathways. Several recent advances offer promise that elucidation of these fundamental issues in reproductive endocrinology are realistically achievable. Two specific aims, based upon emerging concepts of conformational plasticities of both the hormone and receptor, are proposed for this project. 1. Delineation of hCG and LHR binding determinants: This aim represents a continuation of ongoing studies to elucidate structures of and contact sites between wild type and engineered analogs of hCG and the receptor ectodomain, with a rigorous test of the proposed model of the ectodomain that is postulated to have a cusp shape due to a series of leucine-rich repeats. 2. Mechanisms of receptor activation and Gs coupling: This aim addresses the mechanisms by which wild type and engineered extracellular hCG-ectodomain complexes activate the transmembrane portion of the receptor, the resulting structural changes of the transmembrane helices and intracellular loops responsible for LHR activation, and elucidation of determinants in LHR required for Gs activation. A combination of protein engineering, biophysical investigations, and cell/molecular biological techniques will be utilized to address these aims. Success of this project will greatly enhance our understanding of fundamental mechanisms in male and female reproductive endocrinology and provide new information to assist rational design of LHR agonists and antagonists.